Myogenin and MyoD are proteins that bind to the regulatory regions of a battery of skeletal muscle genes and activate their transcription during muscle differentiation. We found that both proteins interact with the enhancer of the nicotinic acetylcholine receptor (nAChR) alpha subunit, a gene that is regulated by innervation. Gel retardation and DNA footprinting were used to identify the regions in the enhancer bound myogenin and MyoD. The regions contained a consensus sequence for the MyoD binding site. The functional role of the binding sites was assessed by transfecting myotubes with a construct containing the a subunit enhancer linked to the gene chloramphenicol acetyl transferase (CAT). Point mutations in the binding sites specifically obliterated expression of CAT in the transfected muscle cells. Since the nAChR gene is regulated by myogenin and MyoD, we decided to study how the expression of these factors is regulated during myogenic differentiation, and during innervation. The levels of myogenin and MyoD transcripts were found to decrease in mouse hind muscle during the perinatal period, a time when innervation is taking place. This decrease is due to innervation, since denervation of adult skeletal muscle lead to a dramatic increase in the levels of both transcripts. Interestingly, the responses of myogenin and MyoD to innervation and denervation, precede the changes in nAChR mRNA levels. Muscle "electrical activity", in absence of nerve, was found to down-regulate the levels of myogenin and MyoD transcripts. Since myogenin and MyoD are trans-regulatory factors that activate the expression of a large battery of skeletal muscle genes during differentiation, it is interesting to speculate that their repression during innervation could lead to the selective down-regulation of synaptic genes. We have also focused in identifying the cis-elements that regulate transcription of the nAChR gamma subunit gene. Two regions that regulate the transcription of the gamma subunit gene during myoblast differentiation were identified. One region enhances the expression of the CAT reporter gene driven by the gamma subunit upstream regulatory sequence, and another site represses its expression. Experiments are in progress to determine if these regulatory sequences function as classical enhancers and silencers, and if they are regulated by myogenic factors related to myogenin or MyoD.